This protocol makes it possible to manipulate proteins, or microRNA expression, in differentiated adipocytes to study their role in adipocytes function. This reverse-transfection method is a simple, economical, and highly efficient method to transfect oligonucleotides into mouse 3T3-L1 adipocytes. This method can also be applied to human preadipocytes differentiated into adipocytes.
Demonstrating the procedure will be Melanie Gaudfrin, a technician from our laboratory. Grow the 3T3-L1 fibroblasts in 100 millimeter dishes in DMEM without pyruvate, 25 millimolar glucose, 10%newborn calf serum, and one percent penicillin and streptomycin. Place the dishes in a tissue culture incubator at 37 degrees Celsius and seven percent carbon dioxide.
Two days after confluence, change the culture medium, replacing it with DMEM without pyruvate, 25 millimolar glucose, 10%FCS, and one percent penicillin and streptomycin supplemented with 0.25 millimolar IBMX, 0.25 micromolar dexamethasone, five micrograms per milliliter insulin, and 10 micromolar rosiglitazone. Incubate the dishes for two days. Two days later, replace the culture medium with DMEM without pyruvate, 25 millimolar glucose, 10%FCS, and one percent penicillin and streptomycin supplemented with five micrograms per milliliter insulin and 10 micromolar rosiglitazone and incubate for another two days.
Feed the cells every two days with DMEM without pyruvate, 25 millimolar glucose, 10%FCS, and one percent penicillin and streptomycin and keep the cells in the incubator. One day or a few hours before the transfection, prepare a solution of collagen type I at 100 micrograms per milliliter in 30%ethanol from a stock solution at one milligram per milliliter. Add 250 microliters of collagen per well of a 12-well plate or 125 microliters per well of a 24-well plate and spread the solution over the surface of the well.
Leave the plate without the lid under the culture hood until the collagen dries. Then wash it twice with D-PBS. Pipette the siRNA with improved minimum essential medium to mix and incubate for five minutes at room temperature.
Add the transfection reagent and the improved minimal essential medium to the siRNA and pipette to mix. Incubate it for 20 minutes at room temperature. Then add the transfection mix to each well of the collagen-coated plate.
Wash the cells in the 100 millimeter Petri dish twice with D-PBS. Add 5x trypsin to the cells, making sure to cover the entire surface with the trypsin. Wait for 30 seconds and carefully remove the trypsin.
Incubate the Petri dish for five to 10 minutes at 37 degrees Celsius in the incubator. Then tap the dish to detach the cells, avoiding cell damage. Add 10 milliliters of DMEM without pyruvate, 25 millimolar glucose, 10%FCS, and one percent penicillin and streptomycin to neutralize the trypsin.
Carefully pipette the medium up and down to detach the cells and homogenize the cell suspension. Count the cells using a Malassez counting chamber or an automated cell counter and adjust the concentration of the cells to 0.625 million cells per milliliter of medium. Seed 800 microliters of the cell suspension per well of a 12-well plate or 400 microliters of the cell suspension per well of a 24-well plate containing the transfection mix.
Incubate the plates in a cell culture incubator and do not disturb them for 24 hours. On the next day, carefully replace the supernatant with fresh DMEM without pyruvate, 25 millimolar glucose, 10%FCS, and one percent penicillin and streptomycin and return the cells to the incubator. Typical time points for detecting target knockdown after siRNA or miR delivery are 24 to 48 hours for mRNA and 48 to 96 hours for protein.
Functional experiments can be performed on transfected adipocytes including insulin signaling, glucose uptake, adipokine secretion, lipolysis, and lipogenesis. The adipocytes have been shown to preserve their morphology after the transfection. Two days after transfection, the adipocytes presented multilocular lipid droplets with lipid content not different between the transfected and non-transfected adipocytes.
The mRNA expression of various differentiation markers was unchanged in transfected cells compared to that in non-transfected adipocytes. The reverse-transfection protocol is efficient as more than 70%of the adipocytes were transfected. Three days after the transfection with siRNA against Perilipin-1, the mRNA level of Perilipin-1 had decreased by 70%and the protein level by 63%Perilipin-1 expression was also analyzed by fluorescence microscopy four days after the transfection and was found to have decreased by 92%compared to its expression in control adipocytes.
The reverse-transfection of adipocytes with microRNA mimicking oligonucleotides to up-regulate the expression of miR-34a is shown here. The over-expression of miR-34a led to the decrease in VAMP2 protein expression by 50%The knockdown of Perilipin-1 in 3T3-L1 adipocytes led to an increase in basal lipolysis while the over-expression of miR-34a led to the inhibition of insulin-induced protein kinase B phosphorylation and glucose uptake. It's important to reach a high level of adipocytes differentiation to perform the transfection on newly differentiated adipocytes and to carefully monitor the treatment of adipocytes with trypsin.
Following the transfection, it is possible to perform functional experiments on the adipocytes to study the impact of proteins or microRNA manipulation on insulin signaling, glucose uptake, lipogenesis, and lipolysis.
